DNA sequencing with reagent recycling on wiregrid

ABSTRACT

The present invention relates to DNA sequencing with reagent cycling on the wiregrid. The sequencing approach suggested with which allows to use a single fluid with no washing steps. Based on strong optical confinement and of excitation light and of cleavage light, the sequencing reaction can be read-out without washing the surface. Stepwise sequencing is achieved by using nucleotides with optically cleavable blocking moieties. After read-out the built in nucleotide is deblocked by cleavage light through the same substrate. This ensures that only bound nucleotides will be unblocked.

This application a continuation of U.S. patent application Ser. No.14/371,890, filed on Oct. 23, 2014, which is the U.S. National Phaseapplication, under 35 U.S.C. § 371 of International Application No.PCT/IB2013/050168, filed on Jan. 9, 2013, which claims the benefit ofU.S. Patent Application No. 61/586,260, filed on Jan. 13, 2012. Theseapplications are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the determination of the sequence ofnucleic acid. In particular, the present invention relates to a devicefor optically controlling an iterative stepwise reaction to determine asequence of a nucleic acid, a method for optically controlling aniterative stepwise reaction to determine a sequence of nucleic acid, aprogram element, a computer-readable medium on which a program elementis stored and the use of a moiety as a blocking moiety in DNAsequencing.

BACKGROUND OF THE INVENTION

DNA sequencing is a rapidly developing field with key players such asIllumina (using the Solexa technology), Life (using the Solidtechnology) and Roche Diagnostics (using the 454 technology). Thedrawback of the sequencing methods these companies use is that thesequence information is obtained by a repetition of several steps whichinvolve replacing reagents and washing. This is cumbersome and timeconsuming and wastes a lot of expensive reagents. The number ofrepetitions is equal to the number of nucleotides that are interrogated,i.e. the read length. With the desire to increase read length thisproblem will become more severe in the future. Currently, this iscompensated by highly parallel sequencing on large arrays. However, forclinical applications this is less desirable. One would rather have afast answer and a lower multiplexing. At the same time the cost per basepair has to come down significantly. Since after every step, i.e. afternucleotide incorporation, the whole surface needs to be washed, allreagents end up in the waste. The total reagent consumption isproportional to the read length and the dominant cost factor ofsequencing at the moment.

The process of sequencing a particular target, such as e.g. DNA becomesmore complex, since the incorporation reaction needs to be followed byan activation reaction and in between careful washing steps arerequired.

Alternative approaches, like that of Pacific Biosciences are moreefficient as they follow the incorporation of nucleotides in real timefor every molecule separately. In this way no washing is required. Theoptical requirements for such a system on the other hand are verysevere, as one needs single fluorophore sensitivity for 4 differentcolors in real time. This can only be achieved for a limited area as thefield of the objective lenses with high magnification is limited forpractical systems and a strong laser light source. With the limited areaonly a small selection of a sample can be sequenced and the risk oferrors due to missed reads is high. The fluorescent signals from labelednucleotides while they are built in by the polymerase need to bediscriminated from those of the same kind of molecules which happen tobe at the same position by chance. This is done by analyzing the pulselength of the fluorescence signal.

SUMMARY OF THE INVENTION

There may be a need to provide an improved determination of the sequenceof a nucleic acid. The present invention matches this need.

The object of the present invention may be seen as to provide for animproved determination of a sequence of a nucleic acid.

The object of the present invention is solved by the subject-matter ofthe independent claims. Further embodiments and advantages of theinvention are incorporated in the dependent claims.

It shall be noted that the herein described embodiments similarlypertain to the method for optically controlling an iterative stepwisereaction to determine a sequence of a nucleic acid, the device foroptically controlling an iterative stepwise reaction to determine thesequence of the nucleic acid, the computer program element, thecomputer-readable medium and the use of a moiety as a blocking moiety inDNA sequencing. Synergistic effects may arise from differentcombinations of the embodiments although they might not be described indetail.

Further on, it shall be noted that all embodiments of the presentinvention concerning a method, might be carried out with the order ofthe steps as described, nevertheless this has not to be the only andessential order of the steps of the method. All different orders andcombinations of the method steps are herewith described.

In the context of the present invention, the term “blocking moiety” isto be understood as a moiety which blocks a synthesizing activity of anenzyme in the case where the blocking moiety is incorporated into amolecule at which the enzyme performs a synthesizing process. A blockingmoiety may be e.g. a blocking molecule.

In the context of the present invention, the term “cleavable” should beunderstood as allowing to be cleaved away by absorbing cleavage light ofwavelength λ_(CL).

In the context of the present invention it should be understood, thatevery embodiment of the optical arrangement disclosed herein may beconfigured to emit polarized excitation light and polarized cleavagelight. Thus, a polarizer or already polarized light sources may be used.Details will be described later on.

Furthermore, the term “excitation light” in the context of the presentinvention applies to the wavelength λ_(Ex1), λ_(Ex2), λ_(Ex3) andλ_(Ex4), respectively.

According to an exemplary embodiment of the invention, a device foroptically controlling a DNA sequence is presented. In particular, thedevice is configured to optically control an iterative stepwise reactionto determine a sequence of a nucleic acid by synthesis. Alternatively,instead of sequencing by synthesis, a synthesis by ligation is also tobe understood in the scope of the present invention. The presenteddevice comprises a substrate for binding at least one molecule on afirst surface of the substrate. The device further comprises an opticalarrangement which is configured to direct excitation light of at least afirst excitation wavelength λ_(Ex1) to the substrate to excite afluorescent label of a first nucleotide which is incorporated into themolecule that is bound on the first surface of the substrate. Theoptical arrangement is further configured to receive and detectfluorescent light emitted by the fluorescent label of the firstnucleotide which is incorporated into the bound molecule. Furthermore,the optical arrangement is configured to direct cleavage light of acleavage wavelength λ_(CL), preferably UV light, to the substrate tooptically induce a photochemical cleavage reaction at the firstincorporated nucleotide to cleave a blocking moiety and the fluorescentlabel away from the first incorporated nucleotide. Furthermore, thesubstrate is configured to confine the excitation light and isconfigured to provide thus for an evanescent wave of the excitationlight as the first surface of the substrate. Furthermore, the substrateis configured to confine the cleavage light, preferably UV light, and isfurther configured to provide for an evanescent wave of cleavage lightat the first surface of the substrate.

Here a device is proposed which is able to combine the advantages ofknown sequencing devices. The devices allows for ensemble based easyread out but no or a reduced number of washing steps is required,meaning a single reagent filling for all reads.

In other words, a sequencing device is presented by the presentinvention, which allows to carry the sequencing out in a single fluidand in which no or a reduced number of washing steps is required. Therequired reagent volume, i.e. costs, is reduced by a factor equal to theread length (50-100). Based on the strong confinement of the excitationlight on the substrate, i.e. a nano-photonic surface structure like awiregrid, the sequencing reaction can be read-out without washing thesurface. Total internal reflection may also be used in order to providethe evanescent wave.

Stepwise sequencing is achieved by using nucleotides with opticallycleavable blocking groups. After read-out, the built-in nucleotide isunblocked by cleavage light like for example UV radiation through thesame nano-photonic substrate. This ensures that only bound nucleotideswill be unblocked. The cost and speed of DNA sequencing is stronglyrelated to reagent consumption. The speed and complexity of thesequencing work stations, instruments and cartridges, is largelydetermined by the necessity of fluid handling for repeating reaction andwashing steps. Both aspects are improved by the present inventionleading to a dramatic exemplification and cost reduction of sequencing.

As it will be explained in detail in the following, the opticalarrangement may also be configured to direct excitation light of afirst, and a second, and a third and a fourth excitation wavelengthλ_(Ex1), λ_(Ex2), λ_(Ex3) and λ_(Ex4), to the substrate to excite afluorescent label of a first nucleotide incorporated into a moleculebound on the first surface of the substrate. Thereby, it can be ensuredthat e.g. four different nucleotides, like for example Adenine (A) andGuanin (G) and Thymine (T) and Cytosine (C), can be distinguished, whenthe respective nucleotide uses a specific and differentiated fluorescentlabel. However, if desired, also only one or two or three of the fourexcitation wavelength λ_(Ex1), λ_(Ex2), λ_(Ex3) and λ_(Ex4) describedabove may be directed by the device towards the substrate to excite themolecule, i.e., the fluorescent label of a nucleotide which isincorporated in the bound molecule. Details about four color systems, inwhich four different fluorescent labels for the above describednucleotides A, G, C, and T are used will be explained hereinafter inmore detail with respect to the following FIGS. 1 and 2. The boundmolecule might be a DNA fragment and can be understood as the nucleicacid whose sequence of nucleotides is determined by the presentinvention.

Furthermore, a person skilled in the art of sequencing or DNA sequencingis aware of the fact that the wavelength λ_(Ex1), λ_(Ex2), λ_(Ex3) andλ_(Ex4) are chosen in combination with the four fluorescent labels usedfor, for example, a nucleotides A, G, C, and T. In other words, thewavelengths are chosen such that the used fluorescent labels, can beoptically excited by the respected excitation light. Furthermore, thewavelength λ_(CL) is chosen such that the desired cleaving reaction ofthe used nucleotides can be optically caused by irradiating saidcleavage light.

As an exemplary embodiment, the following wavelength may be used,although the person skilled in the art may be part from the explicitlydisclosed wavelength. The excitation wavelengths could be chosen basedon the following: Optimal spacing of the excitation wavelengths over thevisible spectrum and in agreement with absorption spectra of mostcommonly used dyes e.g. FAM, HEX, Cy3 Cy5, Alexa Fluor 700 or Atto700,or similar. The excitation wavelengths may also be adjusted toavailability of light sources like e.g. solid-state laser light sourceswith e.g. 405, 532, 633 and 780 nm. However, the invention is notlimited to said excitation wavelengths. The emission maximum of therespective dye may be chosen such that no overlap with the excitationwavelength of the neighboring occurs. Furthermore, the cleavage light,preferably UV light, may be in the range of 250-400 nm, preferably300-370 nm. However, the invention is not limited to said excitationwavelengths.

It shall be noted that the molecule which is bound at the first surfaceof the substrate may for example be a DNA fragment, DNA, RNA, mRNA oranother nucleic acid. Furthermore, also an enzyme, which will bedescribed herein later on, may be bound to the first surface of thesubstrate. In the context of the present invention, the term “bound”shall be understood as a state in which the element is immobilized tothe first surface of the substrate.

In addition, the substrate provides for spots which may be covered withclones of identical molecules, in order to increase the optical signal,which is received by detecting the fluorescence. Therefore, a substratemay be provided as an array of such spots with respectively differentclones, such that throughput of sequencing is increased.

In other words, the above presented device of the present inventionallows for an assembly-based optical sequencing process without washingsteps, such that the total process of determining a sequence of anucleic acid by synthesis can be carried out in a single solution on thesubstrate. The process steps can be controlled optically. The process ofunblocking, which shall be understood in the present invention asactivation, of the incorporated nucleotide can be carried out by theirradiation of a surface selective, evanescent radiation of cleavagelight. Preferably, such irradiation is performed with UV light. Theprocess is described as directing cleavage light of a cleavagewavelength λ_(CL) to the substrate to optically induce the photochemicalcleavage reaction at the first incorporated nucleotide. Therefore, thepresented device is configured to cleave only the fluorescent label awayfrom the first incorporated nucleotide by irradiating the substrate withan evanescent wave of cleavage light. Thus, only incorporatednucleotides will be unblocked, activated or cleaved due to thelocalization of the evanescent field of cleavage light. The sameevanescent field illumination is used by the presented device forreading the fluorescence of the incorporated basis or nucleotidesagainst the background of fluorescent labels in solution. Thelocalization of the optical field of excitation light, which comprisesat least the first excitation wavelength λ_(Ex1), is achieved by theevanescent field of said excitation light.

For example, the evanescent wave of cleavage light and the evanescentwave of excitation light can be generated by the substrate of thepresented device by providing for a wiregrid. This may allow for using afocused beam of high intensity such that the photo-optical reactionoccurs at a high rate in a very limited area very close to the surface.The optical arrangement may comprise respective optical elements for theexcitation and detection of fluorescence, i.e. the read-out, andrespective optical elements for unblocking, i.e. activation, in a singleoptical arrangement unit or may also be comprised in physicallydifferentiated elements.

Furthermore, the respective excitation light source may be comprised bythe optical arrangement. Furthermore, the light source for emittingcleavage light may be comprised by the optical arrangement. Illuminationfor cleaving, i.e. unblocking and read-out, i.e. excitation anddetection of fluorescence, can optionally occur through the same lens.However, if desired, also two different optical set-ups for unblockingand reading-out can be presented.

The evanescent wave field used in the present invention decaysexponentially with distance from the first surface, with a decay lengthdepending on the complex refractive index of the material, e.g. metal,of the wiregrid and that of the medium between the wires, under thecondition that the aperture between the metal wires is smaller than theoptical resolution at the pertinent wavelength. For example the aperturemay be 70 nm which is well below that limit also for UV light. The decaylength is estimated as 16.8 nm (see below), which means that the fieldhas decreased to 1/e of the irradiated intensity at that distance fromthe interface of the metal wire and the substrate. This might beslightly different from the first surface due to underetching of thefirst surface between the wires.

In other words, the presented device provides for a surface selectivecleavage light irradiation and a surface of excitation lightirradiation. The substrate may be configured as a nano-photonic surfacestructure, such that the above described optical confinements of theexcitation light and the cleavage light and additionally the evanescentwaves of cleavage light and excitation light are generated. Thecombination of inter alia, the excitation light, i.e. the read-outoptics, and the cleavage light allow for an iterative stepwise reactionto determine the sequence of nucleic acid by synthesis. This has thebenefit that washing steps can be omitted. Subsequently, the steps andcycles described above which are performed with the presented device,can be repeated many times to allow for a stepwise incorporation of oneor more further nucleotides into the bound molecule and afterwardsreading out, whether said nucleotide has been incorporated or not asdescribed above.

The device may be further configured to achieve data which describe thedifferences of the nucleic acids that were incorporated into the boundmolecule based on the optically stepwise action that is controlledoptically by the device.

Furthermore the substrate may be out of a polymer e.g.poly-(cyclo-)olefin, poly-carbonate, polyester or PMMA. Also metal andsemiconductors may be used.

According to another exemplary embodiment of the invention, the devicefurther comprises the molecule which is bound to the first surface ofthe substrate. The device further comprises a solution with a pluralityof nucleotides and an enzyme. Therein, the nucleotides respectivelycomprise the blocking moiety. The blocking moiety is configured to blocka synthesizing activity of the enzyme when the respective moiety isincorporated into the molecule bound to the first surface of the device.

If desired, the blocking moiety comprises the fluorescent label.However, the blocking moiety and the fluorescent label may beincorporated or positioned at the first nucleotide at differentpositions. They may be cleaved away in one single cleavage process or indifferent cleavage processes. This holds for every embodiment of thepresent invention.

The herein presented embodiment provides for the advantage that thereaction of incorporation stops on its own due to the comprised blockingmoiety. For example, steric hindering can used by the blocking moiety toblock the synthesizing activity of the enzyme. This allows doing a localread-out of a reaction that is occurring on many spots simultaneously.In the prior art the action of used enzymes in a sample in the devicecannot be synchronized. However, the present invention allows for theincorporation of nucleotides, which can advantageously be done step bystep together with a read-out after every step. The advantage isachieved by using the above-described blocking moieties, which may beblocking nucleotides, that block the activity of the enzyme after thenucleotide is incorporated. Active unblocking is required to continuewith the incorporation of the next nucleotide, which the presentinvention allows by the optical arrangement that is configured to directcleavage light to cleave away the blocking moiety. The blocking moietymay comprise the fluorescent label.

As exemplary embodiments, the blocking moieties may be embodied as3′-blocked reversible terminator or as 3′-unblocked reversibleterminator as described and defined in “Sequencing technologies, thenext generation” by Michael L. Metzker, Nature Review Genetics 11 (2010)31-46. Therein, also termed “unblocked”, said blocking moieties3′-unblocked reversible terminator can be used as blocking an activityof an enzyme. Reversible terminators may be understood as ligandsattached to the nucleotide/ribose unit which stop the incorporation ofany subsequent nucleotide after the incorporation. They are reversiblewhen upon cleavage by chemical or photochemical means this process canbe undone and the polymerase can build in the next nucleotide.Furthermore, the 3′-blocked reversible terminator of Metzker et al canbe amended, for example chemically, to make them photo cleavable. Then,the photo cleavage with the cleavage light can be performed by means ofthe present invention. In addition, other complexes may be used asblocking moieties in combination with the respective enzyme as will bedescribed later on. The skilled person knows, which combination ofenzyme and blocking moiety leads to the desired effect of blocking thesynthesizing activity of the enzyme.

The herein used terminology regarding said blocking moieties is in linewith and adapted to the disclosure of said article. Said second class ofmoiety is of an additional advantage as is the case of the 3′-positionof the ribose unit is unblocked and the incorporation of the nextnucleotide is prevented by the bulky group that also contains thefluorescent label attached to the base pairing moiety at 5′-position ofthe ribose unit. This can also be gathered from the following FIG. 6 andits description. Propagation of the nucleotide oligomer is prevented aslong as this group is attached. However, incorporation of the nextnucleotide is enabled after removal of the bulky group. The presentinvention provides for such a removal by inducing a photochemicalcleavage reaction. More advantageously, the present invention uses suchcleavage reaction only very close to the surface at which the moleculeis bound, due to the fact that an evanescent wave of cleavage light isgenerated.

In addition to the following description, the details about FIGS. 6, 8and 10 should be taken into account, in which blocking moieties withcomprised fluorescent label are disclosed.

According to another exemplary embodiment of the present invention, theblocking moiety is a photo-cleavable 3′-unblocked reversible terminator.

According to another exemplary embodiment, the blocking moiety is chosenfrom the group comprising a derivative of nitrophenylethyl,5-methyl-(2-(2-nitrophenyl)propyl)carbonate-dUTP analogue,5-methyl(2-oxo-1,2-diphenylethyl)carbonate-dUTP analogue, and anycombinations thereof.

According to another exemplary embodiment, the substrate is configuredas a wiregrid for the excitation light and for the cleavage light.

The wiregrid may comprise a pattern of metal wires on, for example, aglass substrate. The spacing between the wires acts as a metal-clad slabwaveguide, in which the major contribution comes from two fundamentalmodes. For example, for TE polarized excitation light incident on thewires of the substrate of the present invention the resulting mode inbetween the wires is the evanescent mode, having an exemplary decaylength of 16.8 nanometres for λ=630 nanometre. Therein assuming thewires of the substrate are filled with a medium having a refractiveindex of water, n=1.33. For TM polarized light, the resulting mode for awiregrid is called a propagating mode, having a decay length of 1.2 μmin this example. For example, the wire height may be 60 nanometres in anexample. Therein the TM polarized mode is transmitted with a loss oflight in the order of 10% or less, while the TE polarized mode isevanescently decaying.

A different way to understand the wiregrid is to think of e.g. aluminiumwires as metals which reflect excitation light with polarizationparallel to the wires (TE polarization) and which transmit polarizationorthogonal to the wires (TM polarization). The maximum transmission ofTM polarized light may be higher than 95%. The evanescent field in thecase of incident TE excitation light is depicted in both FIGS. 3a and 3b. The excitation light and the cleavage light irradiated by the opticalarrangement of the present invention may be of such polarization in thisand every other embodiment of the present invention.

A person skilled in the art unambiguously derives from the abovepresented descriptions that the geometrical parameters of the usedwiregrid are to be adapted to the excitation light and the cleavagelight which is irradiated by the optical arrangement. For example, thecondition that the aperture of the wire grid between the wires should besmaller than the optical resolution at the pertinent wavelength, i.e.aperture<<optical resolution˜λ/2 NA. In the context of the followingfigures, the apertures are termed slit-like openings.

The use of the wiregrid substrate of the presented device provides foran extreme optical confinement. In combination with a fast photochemicalcleavage, which is used to decouple the so-called blocking moiety on thenucleotide to prevent continuation of the incorporation of the nextnucleotide, the indicated advantages are realized. The use of thewiregrid has the additional advantage of being largely independent onthe angle in incidence. Therefore, it can be used in combination withfocused beams to achieve a high intensity locally while keeping the restin the dark. In other words, the wiregrid allows to excite and besensitive to only those molecules, for example DNA fragments, that arevery close to the surface in the evanescent field and thus no detectionor effect on any label nucleotide outside the evanescent field iscaused. For example, the evanescent field may elongate about 20nanometres from the first surface of the substrate. This may be the casefor both the excitation light and the cleavage light.

A wiregrid substrate comprises a second surface opposite of the firstsurface and the optical arrangement is configured to irradiate thesecond surface of the substrate with the excitation light and thecleavage light. In other words, the substrates in the opticalarrangement are positioned relative to each other such that the cleavagelight and the excitation light are directly directed towards the secondsurface of the substrate. This may be seen as a backwards radiation ofthe substrate. On the front surface, the first surface, the regular wirestructure is presented by the wiregrid. Between the regular metal wire,i.e. in the spaces between the wiregrid, the molecule, for example DNAfragments, is bound or immobilized.

The term “excitation light” in the context of the present inventionapplies to the wavelength λ_(Ex1), λ_(Ex2), λ_(Ex3) and λ_(Ex4),respectively. Consequently, for all four excitation wavelengths thesubstrate ensures that confinement and a creation of an evanescent waveof the respective light are generated. If desired, more or less lightsources and/or fluorescent labels can be used without departing from thepresent invention.

According to another exemplary embodiment of the invention, the cleavingreaction takes a time t_(cleavage), which depends on an intensity of theirradiated cleavage light. Furthermore, the incorporation of a secondnucleotide into the bound molecule takes a time t_(incorporation). Theherein presented device comprises an optical arrangement which isconfigured and adjusted to provide the irradiated cleavage light with anintensity such that t_(cleavage)<t_(incorporation).

Photo cleavage should only occur in those molecules which areincorporated already and bound to the surface. Reaction in the bulkwould lead to unblocked reagents which could be built in withoutnoticing and in this way introduce errors in the sequencing results.Therefore, it is valuable to only illuminate locally for a short periodto make the cleavage reaction fast compared to the rate of incorporationof nucleotides by the enzyme. Working principle of the enzyme and theblocking moiety has been already described above. That disclosureapplies within the herein described exemplary embodiment. The presentedembodiments allow for synchronizing incorporation of the nextnucleotides and ensure that the detected fluorescent signal is highlyreliable.

The fact that the cleavage light is in an evanescent mode with respectto the substrate provides for the advantage that a repeated exposuredoes not lead to fluorescent labels in the solution which are bleachedand which loose their function. In other words, the presented embodimentavoids such a bleaching and function-loosing of fluorescent labels insolution.

For an improved synchronization of the incorporation of severalnucleotides at several bound molecules, the unblocking step with thecleavage light should be carried out as fast as possible, i.e. with thehighest cleavage light intensity possible. This may be achieved byfocusing the cleavage light, preferably the UV light, with a lens andscanning the surface by moving the lens or the substrate. The unblockingstep may be carried out after reading the sequencing step. This readingcan be carried out by scanning a focus beam or step and scan with fieldillumination. It may also be possible to embody cleavage light as asingle flash of, for example, UV light for the total surface. In view ofthe reaction rate for the base incorporation for the sequencingreaction, the local cleavage light illumination time should be, forexample, below 1 minute.

According to another exemplary embodiment, the substrate comprisesseveral adjacent binding positions for binding molecules for the firstsurface along a first direction. The device is further configured toperform an optical scan by moving the substrate and the opticalarrangement relative to each other along the first direction.Furthermore, the device is configured to perform the optical scan suchthat each binding position is firstly irradiated with the excitationlight of at least the first wavelength λ_(Ex1) and subsequently andsecondly is irradiated with the cleavage light of the cleavagewavelength λ_(CL) in a movement along the first direction.

In other words, a coupling of the cleavage light and the excitationlight is presented which provides additional benefit of simultaneouslyreading-out the fluorescent signals and using the cleavage reaction,such that further nucleotides may be incorporated. This is done in ascanning mode that may provide for high local intensity of the usedelectromagnetic radiation without having the need of high power lightsource. In this arrangement, focused light beams of the cleavage lightand of the excitation light is useful. Therein the unblocking step maybe carried out after the reading the sequencing step. In a preferredembodiment, the read-scanning can be coupled to the unblocking scanningby integrating both light beams in a single actuator. Furthermore, ifdesired, even a single lens might be used aligning the two light beams.Alternative two lenses can be integrated in a single stage or twoseparate stages can operate synchronously. This can also be implementedin the step and scan read approach, in which the cleavage step is alsocarried out in a step and scan mode by irradiating the same region asthe excitation light source.

If desired, the presented device is configured to form such an opticalscan in one continuous movement along the substrate. Therein a repeatedcontinuous scanning is allowed. Thus the device is configured to firstlyread-out whether the first nucleotide is incorporated into the molecule,e.g. a DNA fragment, or not and secondly is configured to cause thephotochemical cleavage reaction at the previously read-out nucleotide incase it is incorporated into the molecule.

According to another exemplary embodiment of the invention, a method foroptically controlling of DNA sequencing, in particular for opticallycontrolling an iterative stepwise reaction to determine a sequence ofnucleic acid by synthesis is presented. The method comprises the stepsof providing a substrate with a molecule bound on a first surface of thesubstrate, irradiating the substrate with the excitation light of atleast a first excitation wavelength λ_(Ex1) by an optical arrangementand thereby optically exciting a fluorescent label of a first nucleotidewhich is incorporated in the bound molecule on the substrate. The methodfurther comprises the step of confining the excitation light by thesubstrate thereby providing for an evanescent wave of the cleavage lightby the substrate of the first surface of the substrate. As a furtherstep, the method defines receiving and detecting fluorescence of theexcited fluorescent label of the first incorporated nucleotide by theoptical arrangement. Furthermore, irradiating the substrate withcleavage light of the cleavage wavelength λ_(CL), preferably UV light,by the optical arrangement and thereby optically inducing aphotochemical cleaving reaction at the first incorporated nucleotide isfurther comprised. A method further defines the step of confining thecleavage light of the cleavage wavelength λ_(CL) by the substratethereby providing for an evanescent wave of the cleavage light by thesubstrate at the first surface of the substrate.

The blocking moiety and the fluorescent labels may be cleaved awaysimultaneously, e.g. in case the blocking moiety comprises thefluorescent label, or may also be cleaved away in different steps.

In other words, the presented method provides for a surface selectiveirradiation of excitation light and of a surface selective irradiationof cleavage light. Consequently, the presented method ensures that onlyfluorescent label incorporated in nucleotides which are incorporated inmolecules bound to the first surface of the substrate, are opticallyexcited to emit a fluorescent signal. Such fluorescent signal of onlyfluorescent label close the first surface are then detected by theoptical arrangement. This enhances the quality of the received anddetected fluorescence signal. Additionally, the presented method ensuresthat only fluorescent labels are cleaved away from nucleotides, whichare incorporated in molecules bound to the first surface of thesubstrate. Herein, a degradation of the solution, or of a moietycomprised therein, which solution is comprised by the device, can beavoided. In other words, the effective concentration of nucleotideswhich can be used for the process of synthesis is not unintentionallydecreased.

Thus, the presented method avoids that fluorescent labels in thesolution are bleached out and loose their function. This may furtherreduce the costs of a process of determining a sequence of a nucleicacid like for example a DNA sequencing.

According to another exemplary embodiment of the invention, a programelement for optically controlling a DNA sequencing, in particular foroptically controlling an iterative stepwise reaction to determine asequence of a nucleic acid by synthesis, which, when being executed by aprocess, be adapted to carry out, irradiating a substrate withexcitation light of at least a first excitation wavelength λ_(Ex1) by anoptical arrangement and thereby optically exciting a fluorescent labelof a first nucleotide which is incorporated into a molecule bound on afirst surface of the substrate, confining the excitation light by thesubstrate thereby providing for an evanescent wave of the excitationlight by the substrate at the first surface of the substrate, receivingand detecting fluorescence of the excited fluorescent label of the firstincorporated nucleotide by the optical arrangement, irradiating thesubstrate with cleavage light of a cleavage wavelength λ_(CL),preferably UV light, by the optical arrangement and thereby opticallyinducing a photochemical cleaving reaction at the first incorporatednucleotide, and confining the cleavage light of the cleavage wavelengthλ_(CL) by the substrate thereby providing for an evanescent wave of thecleavage light by the substrate at the first surface of the substrate,is presented.

According to another exemplary embodiment of the invention, acomputer-readable medium, on which a computer program for opticallycontrolling a DNA sequencing, in particular for optically controlling aniterative stepwise reaction to determine a sequence of a nucleic acid bysynthesis is stored, which, when being executed by a processor, isadapted to carry out irradiating a substrate with excitation light of atleast a first excitation wavelength λ_(Ex1) by an optical arrangementand thereby optically exciting a fluorescent label of a first nucleotidewhich is incorporated into a molecule bound on a first surface of thesubstrate, confining the excitation light by the substrate therebyproviding for an evanescent wave of the excitation light by thesubstrate at the first surface of the substrate, receiving and detectingfluorescence of the excited fluorescent label of the first incorporatednucleotide by the optical arrangement, irradiating the substrate withcleavage light of a cleavage wavelength λ_(CL), preferably UV light, bythe optical arrangement and thereby optically inducing a photochemicalcleaving reaction at the first incorporated nucleotide, and confiningthe cleavage light of the cleavage wavelength λ_(CL) by the substratethereby providing for an evanescent wave of the cleavage light by thesubstrate at the first surface of the substrate, is presented.

The computer program element may be part of a computer program, but itcan also be an entire program by itself. For example, the computerprogram element may be used to update an already existing computerprogram to get to the present invention. The computer-readable mediummay be seen as a storage medium, such as for example a USB stick, a CD,a DVD, a blue ray, a data storage device, a hard disk, or any othermedium, on which a program element as described above can be stored.

It may be seen as a gist of the invention to provide for a new approachfor determining a nucleic acid sequence. Thus sequencing can be carriedout in a single fluid and in which no washing step is required. Based onstrong confinement of firstly excitation light and secondly cleavagelight due to a correspondingly configured substrate like for examplewiregrid, the sequencing reaction can be read-out without washing thesurface. Stepwise sequencing is achieved by using nucleotides withoptically cleavable blocking moieties, that are attached to thenucleotides in the solution, that are to be incorporated in themolecules which are bound to the surface of the substrate. After aread-out with excitation light, which uses fluorescence, the built-in orincorporated nucleotides are unblocked by irradiating cleavage lightthrough the same substrate. This ensures that only bound nucleotides areunblocked. In other words, the method and device of the presentinvention are configured to stepwise and optically induced incorporationof nucleotides with a sequence, which corresponds to a sequence ofnucleotides of the bound molecule. Furthermore, the method and thedevice are configured to stepwise and optically read-out and determinethe sequence of nucleotides which are incorporated in the boundmolecules. Furthermore, the method and the device of the presentinvention are configured to base the determination of the sequence ofthe incorporated nucleotides on the received and detected respectivefluorescence light emitted by the fluorescent label of the respectiveincorporated nucleotides.

According to another exemplary embodiment the use of a moiety as ablocking moiety in DNA sequencing is presented wherein the moiety ischosen from the group comprising a derivative of nitrophenylethyl,5-methyl(2-(2-nitrophenyl)propyl) carbonate-dUTP analogue,5-methyl(2-oxo-1,2-diphenylethyl) carbonate-dUTP analog, and anycombination thereof.

Using the blocking moiety 5-methyl(2-(2-nitrophenyl)propyl)carbonate-dUTP analogues in DNA sequencing device has two advantages.Firstly, it gives defined, less reactive remnants after thephotochemical cleavage resulting in a more clear process. Secondly, ithas a high reaction rate. Compared to other photocleavable molecules,the new molecules are derivatives of the nitrophenylethyl moiety leadingto nitrobenzen derived photoproducts that are much more stable than thenitros compounds generated by photochemistry of the nitrophenylmethylderived molecules. Further more, generation of CO₂ is a driving forceand clean way to efficiently increase the photochemical reaction speed.Another advantage of the use of said blocking moieties in DNA sequencingis demonstrated by the fact that the reaction is completed at very lowcleavage light intensity. This means that the amount of energy neededper spot can be reduced in the DNA sequencing device. Numeral exampleswill be given later on in the context of FIG. 7 and FIG. 9.

These and other features of the invention will become apparent from andare elucidated with reference to the embodiments described hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the invention will be described in thefollowing drawings.

FIG. 1 schematically shows a device according to an exemplary embodimentof the invention.

FIG. 2 schematically shows a device according to an exemplary embodimentof the invention.

FIGS. 3a and 3b schematically show a substrate creating an evanescentwave in the region of the bound molecule used in an exemplary embodimentof the present invention.

FIG. 4 schematically shows a device according to an exemplary embodimentof the invention.

FIG. 5 shows a flow diagram of a method according to an exemplaryembodiment of the invention.

FIG. 6 schematically shows two blocking moieties.

FIG. 7 schematically shows a time course plot of photochemical cleavagerates.

FIG. 8 schematically shows a photochemical cleavage process.

FIG. 9 schematically shows photo transformation graphs.

FIG. 10 shows a photochemical cleavage process of5-methyl(2-oxo-1,2-diphenylethyl) carbonate-dUTP analogs used in anexemplary embodiment of the present invention.

In principle, identical or similar parts are provided with the samereference symbols in the figures.

DETAILED DESCRIPTION OF EMBODIMENTS

FIG. 1 depicts a device 100 for optically controlling an iterativestepwise reaction to determine a sequence of a nucleic acid bysynthesis. The device comprises a substrate 101 for binding at least onemolecule 102 on the first surface 103 of the substrate. The molecule 102which is bound on the first or front surface 103 of the substrate 101can for example be a fragment of a DNA. Furthermore, the opticalarrangement 104 is shown in FIG. 1. FIG. 1 schematically shows that theoptical arrangement is configured to direct excitation light 110 of forexample the first excitation wavelength λ_(Ex1) to the substrate.Furthermore, four different nucleotides are schematically shown and aredepicted with reference signs 109, 116, 117 and 118. For example, afirst nucleotide 109 is shown as Thymine, T. The nucleotide 109comprises a blocking moiety 119. Furthermore, the blocking moiety 119comprises the first fluorescent label 105. In an analog way, secondnucleotide 116 is schematically depicted in FIG. 1, from which can begathered that also a blocking moiety 119 and the second fluorescentlabel 106 is comprised. The third nucleotide 117 comprises also ablocking moiety and a third fluorescent label 107. Additionally, thefourth nucleotide 118 is schematically depicted which comprises also ablocking moiety and a fourth fluorescent label 108. However, sample 114may comprise a much larger plurality of such nucleotides, andnucleotides 109, 116, 117 and 118 are shown here merely as a symbolicdepiction. Furthermore, FIG. 1 shows a solution 114 in which thenucleotides and the enzyme 115 are comprised. In case one of the shownfour nucleotides is incorporated in the bound molecule 102, thepresented device 100 provides for the following advantages. The opticalarrangement is configured to receive and detect fluorescence lightemitted by the fluorescent label of the first nucleotide incorporatedinto the bound molecule 102.

As can further be gathered from FIG. 1, the optical arrangement isconfigured to direct cleavage light 112 of cleavage wavelength λ_(CL) tothe substrate. This allows to optically induce a photochemical cleavagereaction at the first incorporated nucleotide to cleave the respectivefluorescence wave from the first incorporated nucleotide. Furthermore,the substrate 101 is configured to confine excitation light such that anevanescent wave of the excitation light at the first surface of thesubstrate is created. Moreover, the substrate is configured to confinealso the cleavage light such that an evanescent wave of the cleavagelight as the first surface of the substrate is created. This may also beseen in FIGS. 3a and 3b . In the embodiment of FIG. 1, the substrate isconfigured as a wiregrid 113 for the excitation light 110 and for thecleavage light 112. Therefore, the wiregrid 113 comprises a regularpattern, like for example a regular metal wire structure. As can begathered from FIG. 1, slit-like openings are provided between theregular patterns, in which openings the bound molecules 102 areimmobilized at the first surface 103 of the substrate 101. Furthermore,FIG. 1 depicts a processing unit 120 which comprises a computer-readablemedium 121 on which a computer program element 122 is stored. Saidprogram element 122 is adapted to instruct the processing unit 120 tofurther instruct the device 100 to perform the above and below describedmethod for optically controlling an iterative stepwise reaction todetermine a sequence of a nucleic acid by synthesis. The device 100 ofFIG. 1 is configured to stepwise and optically induce the incorporationof nucleotides 109, 116, 117, 119 with a sequence, which iscomplementary to the sequence of nucleotides of the bound molecule 102.In case the molecule 102 is a DNA fragment, the nucleotides comprised bythe sample 114 are incorporated into molecule 102 in a sequence thatcorresponds to the nucleotide sequence of molecule 102.

The device is further configured to base the determination of thesequence of the incorporated nucleotides on the received and detectedresponse fluorescence light emitted by the fluorescent label of therespective incorporated nucleotide. Therefore, the presented device 100of FIG. 1 firstly ensures that only nucleotides are read-out by theexcitation light 110, which nucleotides are incorporated into a boundmolecule 102 by the use of an evanescent wave of the excitation light.Secondly, the device 100 of FIG. 1 ensures that only bound nucleotideswill be unblocked by the cleavage light which avoids unblocking ofnucleotides that are not yet contained i.e. incorporated by the molecule102. Consequently, the detected fluorescence signal 100 may be seen asthe light 111, is highly reliable for the determination of the sequenceof the nucleic acids.

Consequently, the cost and speed of the DNA sequencing performed withthe device 100 of FIG. 1 are both improved. Less fluid is necessary asno washing step is needed. The device of FIG. 1 shows a simplificationand cost reduction of sequencing. The presented device 100 of FIG. 1allows for a new process combination by allowing an assemble-based easyread-out without any washing step, meaning a single reagent filling forall reads. The blocking moieties used within the exemplary nucleotides109, 116, 117, 118 may for example be a photo-cleavable 3′-unblockedreversible terminator. However, also other blocking moieties, using forexample steric hindering, may be used to reach the desired and abovedescribed effects.

Furthermore, the optical arrangement 104 as shown in FIG. 1 may beconfigured to provide the irradiated cleavage light with an intensitysuch that the cleaving reaction time t_(cleavage) is smaller than thetime it takes to incorporate the second nucleotide into the molecule102. As the cleaving reaction time t_(cleavage) depends on the intensityof the irradiated cleavage light, FIG. 1 may provide for a selectedcombination of nucleotides with a specific blocking moiety and aconfiguration of the optical arrangement regarding the intensity of thecleavage light. In other words, the intensity of the cleavage light ofthe device of FIG. 1 is adapted such that for the used combination ofnucleotides and blocking moieties the cleaving reaction timet_(cleavage) is smaller than t_(incorporation).

If desired, additionally or alternatively, the following set-up ofdevice 100 may be provided to the user. The residence may be seen as anaverage residence time and in the spot of cleavage light of anon-incorporated nucleotide. An optical arrangement may further beconfigured to provide the irradiated cleavage light with an intensitysuch that t_(cleavage) is smaller than t_(residence). Consequently, nodegradation of free and unbound nucleotides due to an undesired cleavagereaction happens. Thus, by configuring the device such that t_(cleavage)is smaller than t_(residence) the probability that a non-incorporatednucleotide is affected by cleaving is reduced or eliminated. In otherwords, to avoid cleavage reactions in the bulk the average residencetime of the molecules in the evanescent field of the wiregrid should besmaller or much smaller than the reaction time required for cleavage atthe pertinent intensity. With a depth of the evanescent field of theorder of 25 nm or less and a diffusion coefficient of the nucleotide ofthe order of 1e-10 m2/s the time it takes for the molecule to diffuse inand out the evanescent field can be estimated as: (5e-8m)2/1e-10=25microseconds. Depending on the illumination time required for unblockingthe bound molecules the probability of damage can be derived. Assume anillumination time of 0.1 s this would be 1:4000, with an illuminationtime of 10 ms it would be 1:400, etc.

Likewise the total damage is proportional to the volume fraction in theevanescent field over the total volume of reagent solution. With achamber height of 100 μm the ratio is 1:4000. This means that in theworst case of damaging all molecules in the evanescent field only 0.025%of the molecules will be damaged. With a read length of 100 finally 2.5%of the molecules in solution would be damaged (worst case) which isstill acceptable from a sequencing point of view.

In the following, information for using the device of FIG. 1, as well asthe devices 100 of FIGS. 2 and 4 is provided. For an improvedsynchronization the unblocking step should be carried out as fast aspossible, i.e. with the highest intensity possible. This can be achievedby focusing the UV-light with a lens and scanning the surface by movingthe lens or the substrate. The unblocking step is carried out afterreading the sequencing step. This reading can be carried out by scanninga focused beam or step-and-scan with field illumination. In a preferredembodiment the read scanning can be coupled to the unblocking scanningby integrating both light beams in a single actuator, possibly even in asingle lens by aligning the light beams. Alternatively, two lenses canbe integrated in a single stage or two separate stages can operatesynchronously. This can also be implemented in the step and scan readapproach, in which the UV-step is also carried out in a step and scanmode by illuminating the same field as the reader. The preferredembodiment will depend on the available UV light source and its power.One can also envision a single flash of UV for the total surface ifenough power is available and/or the area of the sequencing surface islimited. In view of the reaction rate for the base incorporation for thesequencing reaction the local UV illumination time should be well below1 minute.

FIG. 2 shows a device 100 which is configured to optically control aniterative stepwise reaction to determine a sequence of a nucleic acid bysynthesis. Similar to FIG. 1, a wiregrid substrate 113 is shown on whicha plurality of molecules 102 are immobilized, i.e. are bound. As can beseen from FIG. 2, a regular pattern 214 provides for slit-like openings215 in which the molecules 202 are bound on the first surface 203. Thesubstrate comprises several adjacent binding positions 209, 210, 211 and212 for binding molecules to the first surface along a first direction213. Said binding positions may be seen as spots which can be coveredwith clones of identical molecules, such that the optical signal, whichis generated, can be increased. The substrate 101 then provides for anarray of such spots, i.e. of such binding positions, with respectivelydifferent clones. This may enhance the throughput. Both devices 100 ofFIGS. 1 and 2 allow a DNA sequencing with only one liquid, therebyavoiding the need to provide for washing steps in which the solutionliquid is changed. Furthermore, the optical arrangement 104 comprisesfive different light sources 201 to 205. The light sources 201 to 204may be seen as excitation light sources in order to provide for fourdifferent excitation wavelength λ_(Ex1) to λ_(Ex4) as describedpreviously. The light source 205 provides for cleavage light with awavelength λ_(CL). For example, the light source 205 may emit UV light.Reference numeral 206 symbolically depicts a switching device whichallows the optical arrangement 104 to switch between the fivewavelengths λ_(Ex1) to λ_(Ex4) and λ_(CL). Furthermore, the lightemitted by at least one of said light sources 201 to 205 is directedtowards the polarization filter 200. Furthermore, a dichroic mirror 207is shown which transmits the emitted light of the light sources 201 to205 towards the substrate 101. After a fluorescent label has beenexcited by an evanescent wave of excitation light (at least one of thewavelengths λ_(Ex1) to λ_(Ex4)), the fluorescence photons emitted by thefluorescent label or labels are directed towards the dichroic mirror 207and are directed towards fluorescence detector 208. As can be seen fromFIG. 2, the optical arrangement 104 may be scanned along the direction213. Consequently, the device 100 of FIG. 2 is configured to perform anoptical scan by moving the substrate 101 and the optical arrangement 104relative to each other along the first direction 213. Consequently, thedevice allows to perform the optical scan such that each bindingposition is firstly irradiated with the excitation light andsubsequently and secondly is irradiated the cleavage light of thecleavage wavelength in a movement along the first direction 213, Theunblocking step, using the cleavage light, can thus be carried out afterreading the fluorescence of the excited incorporated nucleotides.

FIGS. 3a and 3b show a substrate 101 which is embodied as a wiregrid113. Regular pattern 214, which may be embodied as a regular metal wirestructure provides four slit-like openings 215, in which the molecules112 are immobilized. As symbolized by the arrow, the excitation light110 and the cleavage light 112 is directed to the substrate towards thesecond surface of the substrate. The second surface is opposite to thefirst surface 103 at which the molecules are bound. Consequently, thewiregrid is illuminated from the back. Furthermore, FIGS. 3a and 3b showthat several unbounded nucleotides 109 are present in the solution whichmay later on be incorporated into the molecule 102 bound on the firstsurface. FIGS. 3a and 3b depict the evanescent wave 300 between themetal structures with dimensions smaller than the optical resolution atthe wavelength of the light beam. The evanescent wave is depicted byFIG. 3b by brightness gradation which corresponds to field intensitygradation. FIG. 3b shows electromagnetic field strength for a wiregridilluminated with TE polarized light. High brightness indicates a highintensity and a low brightness indicates a low intensity. The hereinpresented substrate can be exemplarily used in the devices of FIGS. 1and 2 as well as in the device of FIG. 4. However, it should be notedthat surface confinement by evanescent waves can be achieved in otherways, which the person skilled in the art knows.

Total internal reflection may also be used in order to provide theevanescent wave in this or in any other embodiment of the invention.

FIG. 4 shows a device 100 for continuously scanning the substrate 101.The device 100 provides for an optical arrangement 104 with a lightsource 400 for emitting excitation light. By means of a colour filter401 which can be rotated different excitation wavelengths may beprovided. Additionally, a cleavage light source 402 is provided. Lightguiding members 403, 404 are presented in order to direct the light tothe respective optical elements 405, 406. As can be seen, separatelenses and optical channels are used for the excitation light and forthe cleavage light. However, if desired, it can also be combined withthe optical paths of both light sources. The optical arrangement 104shown in FIG. 4 allows for a relative movement between the substrate 101and the optical arrangement 104 in the direction of 407.

FIG. 4 may also comprise a dichroic mirror configured to transmitexcitation light and is configured to reflect the fluorescent lightemitted by the used fluorescent labels. Furthermore, the substrate maybe configured to transmit only a first polarization of light and isconfigured to reflect a second polarization of light which isperpendicular polarized to the first polarization. The polarizationfilter is configured to transmit only the first polarization of light.The device of FIG. 4 is configured to generate data which describes thesequences of the nucleic acids that were incorporated into the boundmolecule based on the optically stepwise action that is controlledoptically by the device.

FIG. 5 shows a flow diagram of a method for optically controlling aniterative stepwise reaction to determine a sequence of a nucleic acid bysynthesis. The method comprises the step of providing a substrate with amolecule bound on a first surface of the substrate in step S1. Byirradiating the substrate with excitation light of at least firstexcitation wavelength λ_(Ex1) by an optical arrangement and therebyoptically excited a fluorescent label of a first nucleotide which isincorporated in the bound molecule on the substrate, is shown with stepS2. Furthermore, step S3 depicts the step of confining the excitationlight by the substrate thereby providing for an evanescent wave of thecleavage light by the substrate at the first surface of the substrate.Receiving and detecting fluorescence of the excited fluorescent label ofthe first incorporated nucleotide by the optical arrangement ispresented by step S4. Step S5 irradiating the substrate with cleavagelight of the cleavage wavelength λ_(CL), preferably UV light, by theoptical arrangement and thereby optically inducing a photochemicalcleaving reaction at the first incorporated nucleotide, is depicted withstep S5. Furthermore, in step S6 confining the cleavage light of thecleavage wavelength λ_(CL) by the substrate thereby providing evanescentwave of the cleavage light by the substrate at the first surface of thesubstrate is provided.

By repeating the presented method steps S1 to S6 the user is enabled todetermine the sequence of nucleic acid that have been incorporated intoa molecule bound to the first surface of the substrate. Consequently,after steps S1 to S6 the user may perform, if desired, the followingsteps. Incorporating a second nucleotide into the molecule bound at thefirst surface of the substrate; then blocking an activity of an enzymeby the second nucleotide after its incorporation the molecule.Irradiating the substrate with excitation light by the opticalarrangement and thereby optically exciting the fluorescent label of thesecond incorporated nucleotide may be performed as well. Confining theexcitation light by the substrate, thereby providing evanescent wave ofthe excitation light by the substrate at the first surface of thesubstrate is a further step of this secondary cycle. The step ofreceiving and detecting fluorescence of the excited fluorescent label ofthe second incorporated nucleotide may then be performed. Irradiatingthe substrate with cleavage light, preferably UV light, by the opticalarrangement and thereby optically and using a photochemical cleavingreaction at the second incorporated nucleotide can be performed as well.As another step for confining the cleavage light by the substratethereby providing for an evanescent wave of the cleavage light by thesubstrate at the first surface of the substrate is presented.

FIG. 6 shows blocking moieties of “Sequencing technologies, the nextgeneration” by Michael L. Metzker, Nature Genetics 11 (2010) 31.Firstly, by using a 3′-blocked reversible terminators and secondly byusing a 3′-unblocked reversible terminators. The 2nd class is veryinteresting as in this case the 3′ position of the ribose unit isunblocked and the in-corporation of the next nucleotide is prevented bythe bulky group that also contains the fluorescent label attached to thebase paring moiety at 5′ position of the ribose unit as can be seen inFIG. 6.

The present invention may use wiregrid technology and the un-blockingstep is done with e.g. polarized UV light of 365 nm. Consequently, afterthis unblocking step the next labeled nucleotide is built in anddetected by scanning the wiregrid using polarized light such that onlythe labeled nucleotides at the DNA fragment at the surface are detected.After this is done, again by providing an unblocking step using UV lightthe next labeled nucleotide can be built in and detected etc. For thisprocess to work one may need: 1. A photo-cleavable 3′-unblockedreversible terminator, as with the 3′ blocked variants the removal ofthe 3-blocking groups (—N3 or —CH2) have to be done in phase. 2. Thephoto-cleaving reaction should be faster than the incorporation of newnucleotides by the polymerase. The so-called 3′-OH unblocked terminatorsinvented by Metzker et al, namely: 2-nitrobenzyl alkylated HOMedUtri-phosphates might be slow for this purpose compared to blockingmoieties we present later on, see FIGS. 8 to 10.

FIG. 7 shows a time course plot of photochemical cleavage rates ofdU.I-dU.V incorporated into the BODIPY-FL labelledprimer-1/oligoTemplate-4 complex using Terminator polymerase. FIG. 7 istaken from V. A. Litosh, W. Wu, B. P. Stupi, J. Wang, S. E. Morris, M.N. Hersh, and M. L. Metzker, “Improved nucleotide selectivity andtermination of 3′-OH unblocked terminators by molecular tuning of2-nitrobenzyl alkylated HOMedU triphosphates”, Nucl. Acids Res, Vol 36,issue 6, 2011, E39. As can be clearly see from this FIG. 7 the timescale at which the photo-cleavage effect occurs is in the orders of 10s. Or as concluded by these authors: “All5-(2-nitrobenzyloxyy)methyl-dUTP analogues were photo-chemically cleavedto 100% efficiency within 60 s at 365 nm UV light exposure with anintensity of ˜0.7 W/cm² in azide solution”. Importantly these authorsalso found that the Terminator polymerase continued to show goodactivity even after being exposed to 365 nm UV light for 150 min withintensities up to 1 W/cm2.

However this photo-cleave chemistry is slower to what we found withdifferent blocking moieties we used. Our approach might improve thenon-cycling reaction we propose. Note the light intensities needed forin the chemistry by Metzker et al is 1 W/cm2 which translates to 10nW/(μm)2. We have used a chemistry using a moiety as a blocking moietyin DNA sequencing, wherein the moiety is a derivative ofnitrophenylethyl. For example, 5-methyl(2-(2-nitrophenyl)propyl)carbonate-dUTP analogues, which have two advantages. First, it givesdefined, less reactive remnants after the photochemical cleavageresulting in a more clear process. Second, it has a higher reaction rateas we have determined independently (see FIG. 9).

Compared to the photocleavable molecules described by Metzger, the newmolecules are derivatives of the nitrophenylethyl moiety leading tonitrobenzen derived photoproducts that are much stable than the nitroscompounds generated by photochemistry of the nitrophenylmethyl derivedmolecules of Metzger. Further more, generation of CO2 is a driving forceand clean way to efficiently increase the photochemical reaction speed.One further advantage of our chemistry is demonstrated by the fact thatthe photochemical cleavage reaction of compound 101 that is from aphotochemical point of view very similar to the molecule of FIG. 8,completes within 30 minutes using a PL10 lamp at 10 cm which produces 4mW/cm2. Compound 101 is depicted in FIG. 9 at the bottom. Also for thedata see FIG. 9. Thus the reaction is completed slower as the example ofthe compounds of the reference cited above but, at 250× lower intensity.This means that the amount of energy needed per spot is 50 times lower.

FIG. 9 shows the efficiency of photo-transformation under 365 nm lightof the compound 101 (our data). Note the spectra changes after 1 h of UVabsorption blue to pink spectrum. B) Graph of the evolution of thephotochemical reaction by the rise of the spectral peak at 319 nm as afunction of excitation at 365 nm. Note that this data in ethanol showsthat the evolution is complete after 30 min. (and for 80% complete after20 min) by excitation with a PL10 lamp at 10 cm which is equivalent toan energy of 4 mW/cm2.

A possible alternative for the nitro-compounds are the“5-methyl(2-oxo-1,2-diphenylethyl) carbonate-dUTP analogs” shown in FIG.10 which exhibit also an efficient photochemical cleavage. They mightalso be used as a blocking moiety in any embodiment of the presentinvention, if desired.

Furthermore, the following compound may be used as blocking moietyaccording to the present invention:

The following variations may also be used as blocking moiety accordingto the present invention: Compound 1 with: X═O(CH2)nZ with n=integerfrom 1 till 18 or X═O(C2H4O)nCH2Z with n=integer from 1 till 20 with Z═Hor a linker connected to a fluorescent moiety and Y═(CH2)nA withn=integer from 0 till 18 or Y═O(CH2)nA with n=integer from 1 till 18 orO(C2H4O)nCH2A with n=integer from 1 till 20 with A=H or a linkerconnected to a fluorescent moiety, in such a combination that at leastone of the groups A or Z have a linker connected to a fluorescentmoiety.

Furthermore, the following compound may be used as blocking moietyaccording to the present invention:

With X and Y are independently (CH2)nZ with n=integer from 1 till 18 or(C2H4O)nCH2Z with n=integer from 1 till 20 with Z═H or a linkerconnected to a fluorescent moiety in such combination that least one ofthe groups X or Y has a group Z that has a linker connected to afluorescent moiety.

Other variations to the disclosed embodiments can be understood andeffected by those skilled in the art in practicing the claimedinvention, from the study of the drawings, the disclosure, and theappended claims. In the claims the word “comprising” does not excludeother elements or steps and the indefinite article “a” or “an” does notexclude a plurality. A single processor or other unit may fulfill thefunctions of several items or steps recited in the claims. The mere factthat certain measures are recited in mutually different dependent claimsdoes not indicate that a combination of these measures cannot be used toadvantage. A computer program may be stored/distributed on a suitablemedium such as an optical storage medium or a solid-state mediumsupplied together with or as part of other hardware, but may also bedistributed in other forms, such as via the Internet or other wired orwireless telecommunication systems. Any reference signs in the claimsshould not be construed as limiting the scope of the claims.

The invention claimed is:
 1. A device for optically controlling aniterative stepwise reaction to determine a sequence of a nucleic acid bysynthesis, the device comprising: a substrate for binding at least onemolecule on a first surface of the substrate; and an optical arrangementcomprising a filter disposed on said first surface, wherein the opticalarrangement is configured to direct excitation light of at least a firstexcitation wavelength λ_(Ex1) to the substrate to excite a fluorescentlabel of a first nucleotide, wherein the first nucleotide isincorporated into the molecule bound on the first surface of thesubstrate, wherein the optical arrangement is configured to receive anddetect fluorescent light emitted by the fluorescent label of the firstnucleotide, wherein the optical arrangement is configured to directcleavage light of a cleavage wavelength λ_(CL) to the substrate tooptically induce a cleavage reaction at the first nucleotide to cleave ablocking moiety and the fluorescent label away from the firstnucleotide, wherein the substrate is configured to provide for anevanescent wave of the excitation light at the first surface of thesubstrate, wherein at least one of the excitation light or the cleavagelight is polarized light, wherein said filter is configured to reflectsaid at least one of the excitation light or the cleavage light and totransmit light having a polarization that is different from apolarization of said at least one of the excitation light or thecleavage light.
 2. The device according to claim 1, the device furthercomprising: a solution with a plurality of nucleotides and an enzyme,wherein the nucleotides of the solution respectively comprise arespective blocking moiety, wherein the respective blocking moiety isconfigured to block a synthesizing activity of the enzyme when therespective nucleotide is incorporated into the molecule bound to thefirst surface.
 3. The device according to claim 2, wherein the blockingmoiety is a photo cleavable 3′-unblocked reversible terminator.
 4. Thedevice according to claim 2, wherein the blocking moiety is selectedfrom the group consisting of a derivative of nitrophenylethyl,5-methyl(2-(2-nitrophenyl)propyl) carbonate-dUTP analogue,5-methyl(2-oxo-1,2-diphenylethyl) carbonate-dUTP analog, and anycombination thereof.
 5. The device according to claim 1, wherein thecleavage reaction takes a time t_(cleavage), wherein the cleavagereaction time t_(cleavage) depends on an intensity of the irradiatedcleavage light, wherein incorporating a second nucleotide into the boundmolecule takes a time t_(incorporation), and wherein the opticalarrangement is configured to provide the irradiated cleavage light withan intensity such that t_(cleavage)<t_(incorporation).
 6. The deviceaccording to claim 1, wherein the substrate comprises several adjacentbinding positions for binding molecules to the first surface along afirst direction, wherein the device is configured to perform an opticalscan by implementing a relative movement between the substrate and theoptical arrangement, and wherein the device is configured to perform theoptical scan such that each binding position is firstly irradiated withthe excitation light of at least the first wavelength λ_(Ex1) andsubsequently and secondly irradiated with the cleavage light of thecleavage wavelength λ_(CL) in a movement along the first direction. 7.The device according to claim 1, wherein the device is configured tostepwisely and optically induce the incorporation into the boundmolecule of nucleotides with a sequence, which is complementary to asequence of nucleotides of the bound molecule, wherein the device isconfigured to stepwisely and optically read out and determine thesequence of nucleotides which are incorporated into the bound molecule,and wherein the device is configured to base the determination of thesequence of the incorporated nucleotides on the received and detectedrespective fluorescent light emitted by the fluorescent label of therespective incorporated nucleotide.
 8. The device according to claim 1,wherein the substrate is configured to confine the excitation light,wherein the substrate is configured to confine the cleavage light, andwherein the substrate is configured to provide for an evanescent wave ofcleavage light at the first surface of the substrate.
 9. A method foroptically controlling an iterative stepwise reaction to determine asequence of a nucleic acid by synthesis, the method comprising:providing a substrate with a molecule bound on a first surface of thesubstrate, wherein a filter is disposed on said first surface;irradiating the substrate with excitation light of at least a firstexcitation wavelength λ_(Ex1) by an optical arrangement and therebyoptically exciting a fluorescent label of a first nucleotide, whereinthe first nucleotide is incorporated in the bound molecule on thesubstrate; providing an evanescent wave of the excitation light by thesubstrate at the first surface of the substrate; receiving and detectingfluorescence of the excited fluorescent label of the first nucleotide bythe optical arrangement; irradiating the substrate with cleavage lightof a cleavage wavelength λ_(CL) by the optical arrangement and therebyoptically inducing a cleaving reaction at the first incorporatednucleotide, wherein at least one of the excitation light or the cleavagelight is polarized light; and reflecting said at least one of theexcitation light or the cleavage light by said filter, wherein thefilter is configured to transmit light having a polarization that isdifferent from a polarization of said at least one of the excitationlight or the cleavage light.
 10. The method according to claim 9, themethod further comprising: providing a solution with a plurality ofnucleotides and an enzyme, wherein the nucleotides of the solutionrespectively comprise a respective blocking moiety; and blocking asynthesizing activity of the enzyme by the respective blocking moietywhen the respective nucleotide is incorporated into the molecule boundto the first surface, wherein the cleaving reaction is performed suchthat the respective blocking moiety is cleaved away from the respectivenucleotide.
 11. The method according to claim 9, wherein the blockingmoiety is selected from the group consisting of a derivative ofnitrophenylethyl, 5-methyl(2-(2-nitrophenyl)propyl) carbonate-dUTPanalogue, 5-methyl(2-oxo-1,2-diphenylethyl) carbonate-dUTP analog, andany combination thereof.
 12. The method according to claim 9, whereinthe substrate comprises several adjacent molecule binding positions atwhich a molecule is respectively bound to the first surface along afirst direction, the method further comprising: performing an opticalscan by implementing a relative movement between the substrate and theoptical arrangement, wherein the optical scan is performed such thateach bound molecule is firstly irradiated with the excitation light ofat least the first excitation wavelength λ_(Ex1) and subsequently andsecondly irradiated with the cleavage light of the cleavage wavelengthλ_(CL) in a movement along the first direction.
 13. The method accordingto claim 12, wherein the cleaving reaction takes a time t_(cleavage),wherein the cleaving reaction time t_(cleavage) depends on an intensityof the irradiated cleavage light, the method further comprising:incorporating a second nucleotide into the bound molecule, wherein theincorporation takes a time t_(incorporation); and selecting theintensity of the irradiated cleavage light at the optical arrangementsuch that t_(cleavage)<t_(incorporation).
 14. The method according toclaim 9, further comprising: confining the excitation light by thesubstrate; and confining the cleavage light by the substrate therebyproviding for an evanescent wave of the cleavage light by the substrateat the first surface of the substrate.
 15. A program element foroptically controlling an iterative stepwise reaction to determine asequence of a nucleic acid by synthesis according to the method of claim9, which, when being executed by a processor, is adapted to carry out:irradiating a substrate with excitation light of at least a firstexcitation wavelength λ_(Ex1) by an optical arrangement and therebyoptically exciting a fluorescent label of a first nucleotide, whereinthe first nucleotide is incorporated into a molecule bound on a firstsurface of the substrate and wherein a filter is disposed on said firstsurface; providing for an evanescent wave of the excitation light by thesubstrate at the first surface of the substrate; receiving and detectingfluorescence of the excited fluorescent label of the first nucleotide bythe optical arrangement; irradiating the substrate with cleavage lightof a cleavage wavelength λ_(CL) by the optical arrangement and therebyoptically inducing a cleaving reaction at the first nucleotide, whereinat least one of the excitation light or the cleavage light is polarizedlight; and reflecting said at least one of the excitation light or thecleavage light by said filter, wherein the filter is configured totransmit light having a polarization that is different from apolarization of said at least one of the excitation light or thecleavage light.
 16. A non-transitory computer-readable medium, on whicha computer program for optically controlling an iterative stepwisereaction to determine a sequence of a nucleic acid by synthesisaccording to the method of claim 9 is stored, which, when being executedby a processor, is adapted to carry out: irradiating a substrate withexcitation light of at least a first excitation wavelength λ_(Ex1) by anoptical arrangement and thereby optically exciting a fluorescent labelof a first nucleotide, wherein the first nucleotide is incorporated intoa molecule bound on a first surface of the substrate and wherein afilter is disposed on said first surface; providing an evanescent waveof the excitation light by the substrate at the first surface of thesubstrate; receiving and detecting fluorescence of the excitedfluorescent label of the first nucleotide by the optical arrangement;and irradiating the substrate with cleavage light of a cleavagewavelength λ_(CL) by the optical arrangement and thereby opticallyinducing a cleaving reaction at the first nucleotide, wherein at leastone of the excitation light or the cleavage light is polarized light;and reflecting said at least one of the excitation light or the cleavagelight by said filter, wherein the filter is configured to transmit lighthaving a polarization that is different from a polarization of said atleast one of the excitation light or the cleavage light.